Development of Systemic in vitro Evolution and Its Application to Generation of Peptide-Aptamer-Based Inhibitors of Cathepsin E

Koichiro Kitamura, Chuya Yoshida, Yasunori Kinoshita, Tomoko Kadowaki, Yoko Takahashi, Takahiro Tayama, Tomoyo Kawakubo, Mohammed Naimuddin, Md Salimullah, Naoto Nemoto, Kazunori Hanada, Yuzuru Husimi, Kenji Yamamoto, Koichi Nishigaki

Research output: Contribution to journalArticlepeer-review

25 Citations (Scopus)


Proteases are involved in various biological functions. Thus, inhibition of their activities is scientifically interesting and medically important. However, there is no systematic method established to date to generate endopeptidase inhibitory peptides. Here, we report a general system to identify endopeptidase inhibitory peptides based on the use of in vitro evolution. Using this system, we generated peptides that inhibit cathepsin E (CE) specifically at a submicromolar IC50. This system generates protease inhibitor peptides utilizing techniques of cDNA display, selection-by-function, Y-ligation-based block shuffling, and others. We further demonstrated the importance and effectiveness of a secondary library for obtaining small-sized and active peptides. CE inhibitory peptides generated by this method were characterized by a small size (8 to 12 aa) and quite different sequences, suggesting that they bind to different sites on CE. Typical CE inhibitory peptide aptamers obtained here (Pi101; SCGG IIII SCIA) have half an inhibition activity (Ki; 5 nM) of pepstatin A (potent CE inhibitor) without inhibiting cathepsin D (structurally similar to CE). The general applicability of this system suggests that it may be useful to identify inhibitory peptides for various kinds of proteases and that it may therefore contribute to protein science and drug discovery. The peptide binding to a protein is discussed in comparison with the antibody binding to an antigen.

Original languageEnglish
Pages (from-to)1186-1198
Number of pages13
JournalJournal of Molecular Biology
Issue number5
Publication statusPublished - Apr 17 2009

All Science Journal Classification (ASJC) codes

  • Structural Biology
  • Molecular Biology


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